The goals of the proposed studies are to elucidate the molecular properties and mechanisms of actions of the kappa 1 (U-69, 593-sensitive) and kappa 2 (U-69,593-insensitive) subtypes of the kappa receptor and provide information about the molecular bases of kappa receptor heterogeneity. Kappa receptors mediate the physiological actions of dynorphin-related peptides and the antinociceptive and sedative actions of ketocyclazocine-like opioids. The proposed research will test the hypotheses that kappa 1 and kappa 2 receptor subtypes mediate excitatory and inhibitory actions of kappa receptor ligands upon neurons in analogy to the M1 and M2 subtypes of the muscarinic receptor. Specifically, experiments are proposed to characterize pharmacologically kappa 1 and kappa 2 opioid receptors of guinea pig and rat brain, determine their localization throughout the brain and upon specific neuronal elements, characterize electrophysiologically the functions which they mediate, and construct anti-idiotypic antibodies against them. Detailed pharmacological analyses using in vitro receptor assays together with electrical recording methods will be used to determine the electrophysiological responses associated with kappa 1 and kappa 2 receptor activation in cultures of embryonic rat striatal and hippocampal neurons. Striatal neurons are particularly suitable because they have been shown to have both kappa 1 and kappa 2 receptor subtypes, whereas rat hippocampal neurons provide a pure source of kappa 2 receptors. Quantitative in vitro autoradiography at the level of the light microscope will be used together with selective neurotoxins to determine upon which neuronal elements kappa 1 and kappa 2 receptors reside in adult rat brain. Monoclonal anti-idiotypic antibodies will be generated for use as tools in kappa receptor physiology and localization. Specifically, the antibodies are important for 1) the determination of regions of homology in the subtypes, 2) determination of the neuroanatomical localization of the kappa 1 and kappa 2 receptors at a high level of resolution, and 3) definitive identification of the functions associated with each of the receptor subtypes as measured electrophysiologically. The overall significance of the proposed research lies in its ability to elucidate the molecular basis of kappa receptor heterogeneity and provide information about kappa 1 and kappa 2 receptor function and localization. These studies are expected to contribute significantly to our understanding of neuronal specialization in the opioid system, of how the brain works with regard to pain perception, and how, on the molecular level, opiates exert their characteristic effects including tolerance, dependence, and addiction.
